Babesia bovis is the major causative agent of tick fever morbidity in domestic cattle ranged on tropical and subtropical pastures. Early work on the immunity conferred on infected animals which survived indicated that the immune response was humoral, since immunity was conferred to calves by colostrum immunoglobulins from infected mothers.
Unresolved antibodies from animals immune to B. bovis were subsequently shown to elicit immunity when transferred to non-immune animals.
Hitherto vaocines have been produced which protect cattle against the severe clinical manifestations of B. bovis parasitaemia. The vaccines used to date include those using killed B. bovis, live vaccines using B. bovis attenuated by either irradiation or rapid passage in splenectomized calves and vaccines derived from in vitro culture supernatant antigens. Although such vaccines are to some extent protective, they all have the inherent problem of containing many antigens. Vaccination with such vaccines therefore elicits an immune response which places the immune system of the vaccinated animal under considerable stress. Also, multi-antigenic vaocines may contain antigens which elicit a strong response activity but which are not especially proteotive due to the location or chemistry of the antigen in the virulent field strains. This strong response may mask or otherwise inhibit the development of immunity based on antibody response to the presence of a less reactive antigen capable in isolation of conferring protective immunity.
Where live attenuated vaccines are used, contamination of the vaccine with other pathogens may occur. Animals vaccinated with the live vaccines are subclinically infected carriers of the parasite and the potential for breakthrough infection due to de-attenuation is present. Refrigerated vaccine has a shelf life of only seven days.
In order to identify specific babesial antigens whioh confer protective immunity, babesial proteins from infected erythrocytes have been fractionated and assayed for immunoprotective potential. Protective antigens have been found in the soluble protein fraction obtained from lysis of babesia-infected erythrocytes as described in Australian patent specification No. 553779. Monoclonal antibodies have been raised against the protective soluble babesia protein fractions, and IFA, ELISA and Western blotting techniques used to identify clones producing monoclonal antibodies against specific babesial antigens. Antigens were then afiinity purified with the monoclonal antibodies to yield single antigens for vaccination testing.
Whilst purified antigen may be produced from babesial lysates by using the monoclonal antibody to that antigen, this method is not suitable for large scale production of vaccination grade product. The requirement therefore arises for improved commercial scale processes for the manufacture of antigen and polypeptides having like immunogenic activity. Attention has therefore been focused on the use of recombinant DNA techniques to transform nonbabesial hosts with babesial genes.
Kemp et al. (Mol. Biochem. Parasit., 12 (1984) 61-67) discloses that babesia genes can transform the eukaryotic yeast host Saccharomyces cerevisiae, and that the transformed host produces poly A.sup.+ RNAs corresponding to the transforming genes. However, there is no data to indicate that the polypeptides corresponding to the poly A.sup.+ mRNA obtained from K-strain B. bovis-infected erythrocytes were protective. The library was amplified in .lambda.-gt10, cleaved, radiolabelled, fractionated, and re-ligated into an ampicillin-resistance-conferring expression vector .lambda.-amp3. Phage were packaged in vitro and plated out on E. coli BTA282 on ampicillin-containing media, and colonies producing cDNA detected by colony hybridization. Colonies expressing babesia antigens were detected by autoradiography using bovine antiserum to K.sub.A strain B. bovis and .sup.125 I-labelled anti-bovine Ig. One clone was selected for further study (designated K.sub.A Bb1). This clone produced a fusion protein of .beta.-galactosidase and a babesial antigen 5-10 kDa larger than native .beta.-galactosidase.
Anti-K.sub.A antisera were fractionated against the fusion protein to yield affinity-purified anti-K.sub.A Bb1 antibodies. These antibodies were used to detect a 220 kDa antigen, corresponding to the dominant 220 kDa antigen detected by unfractionated serum. Immunofluorescent assay indicated that anti-K.sub.A Bb1 reacts only with the Babesia parasite and not with the surrounding erythrocytic cytoplasm in vitro. By contrast, unfractionated anti-K.sub.A serum reacts with both the infected erythrocyte cytoplasm and the parasite.
However, vaocines formulated from the K.sub.A Bb1/.beta.-galactosidase fusion protein did not exhibit significant protective immunization of animals against virulent heterologous B. bovis Challenge (Timms et al., unpublished results, Animal Research Institute, Queensland Department of Primary Industries). Indeed, the results indicated that the K.sub.A Bb1/.beta.-galactosidase vaccines were less efficacious than live K.sub.A vaccines.
The whole approach adopted by Kemp in regard to location of polypeptides protective against babesiosis however was a "shotgun" approach and was not based on an approach which could be used to provide protective native as well as recombinant polypeptides.
Reference also may be made to Cowman described in Manipulation and Expression of Genes in Eukaryotes (1983) 185-188 wherein a gene coding for avirulence - associated polypeptides of B. bovis was isolated by differential colony hybridization.
Poly A.sup.+ RNA was isolated from K-avirulent (K-A) strain by extraction from parasitized cattle erythrocytes and oligo-dT cellulose chromatography. The RNA was copied into double-stranded cDNA which was inserted in pBR322. Transformed cells were plated on nitrocellulose filters and replica filters were hybridized with labelled cDNA probes synthesized from poly(A) RNA of K-A and the virulent K geographical isolate (K-V). cDNA plasmids were selected representing RNAs of differing abundance. Increased levels of RNA molecule were found to correlate with avirulence.
However, the main thrust of this reference was to identify cDNAs corresponding to RNAs which hybridize in greater abundance with probes synthesized from poly A.sup.+ mRNA of the avirulent Ka strain. This reference did not disclose however preparation of polypeptides.